RESUMEN
<p><b>OBJECTIVE</b>To construct a replication-incompetent recombinant adenovirus mediating short hairpin RNA (shRNA)-induced tissue factor gene silencing in the islet.</p><p><b>METHODS</b>Four pairs of complementary oligonucleotides were designed and synthesized to create double-stranded oligonucleotides (ds oligo). The ds oligos were cloned into Pentr/U6 vector to construct the shuttle plasmid pENTR/U6-shRNA, which was transduced into human islets via liposome after sequence verification. The plasmid with the best silencing effect was identified by real-time RT-PCR, followed by homologous recombination with the adenovirus backbone plasmid. The functional clone was transfected into 293A cells to amplify the adenovirus, whose silencing effect against TF expression was tested using real-time RT-PCR and Western blotting.</p><p><b>RESULTS</b>The pENTR/U6-shRNA shuttle plasmid was constructed and verified by sequencing. The recombinant adenovirus-mediated shRNA against TF was constructed, and real-time RT-PCR and Western blotting demonstrated that the strongest silencing effect of the adenovirus against TF occurred on the 4th day following islet transfection.</p><p><b>CONCLUSION</b>Replication-incompetent recombinant adenovirus-mediated shRNA against TF has been successfully constructed, which has good silencing effect against TF expression in human islet in vitro.</p>
Asunto(s)
Humanos , Adenoviridae , Genética , Fisiología , Secuencia de Bases , Línea Celular , ADN Recombinante , Genética , Expresión Génica , Ingeniería Genética , Métodos , Secuencias Invertidas Repetidas , Islotes Pancreáticos , Metabolismo , Plásmidos , Genética , Interferencia de ARN , ARN Interferente Pequeño , Genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tromboplastina , Genética , Carga Viral , Replicación ViralRESUMEN
<p><b>OBJECTIVE</b>To establish a stable method for obtaining large quantity of highly purified immature dendritic cells (imDCs) in vitro, and identify the morphology, function and surface markers of the cells.</p><p><b>METHODS</b>CD117(+) hemopoietic stem cells (HSCs) were isolated and purified from the bone marrow of healthy C57 mice by magnetic affinity cell sorting. After cell expansion by treatment with stem cell factor (SCF) and interleukin-3 (IL-3), the HSCs were induced for directional differentiation into imDCs by treatment with GM-CSF, IL-4 and IL-10. The imDCs obtained were identified by morphological and functional observation under inverted microscope, scanning electron microscope and transmission electron microscope, followed by detection of the expressions of the surface markers using flow cytometry.</p><p><b>RESULTS</b>After 3, 5 and 7 days of culture in the presence of SCF+IL-3, the cells were expanded by 10.34-/+1.43, 22.65-/+2.71 and 54.39-/+3.08 folds, respectively. The HSCs were successfully induced to differentiate into imDCs with phagocytotic activity. The dendrites of the imDCs were short small, and appearing spinous. The expressions of surface markers were detected from the cells showing the phenotype of CD11c(+), I-A/I-E(low), CD40(-), CD80(-), CD86(-).</p><p><b>CONCLUSION</b>The method described allows steadily acquisition of large quanty of highly purified imDCs and of their effective identification in vitro.</p>
Asunto(s)
Animales , Ratones , Técnicas de Cultivo de Célula , Métodos , Diferenciación Celular , Separación Celular , Métodos , Células Cultivadas , Células Dendríticas , Biología Celular , Citometría de Flujo , Células Madre Hematopoyéticas , Biología Celular , Ratones Endogámicos C57BL , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Proteínas Proto-Oncogénicas c-kitRESUMEN
<p><b>OBJECTIVE</b>To investigate the role of simultaneous blockade of CD40/CD40L and B7/CD28 pathways in the immune tolerance via co-expression of sCD40LIg and CTLA4Ig mediated by replication-defective adenovirus.</p><p><b>METHODS</b>Ad-sCD40LIg-IRES(2)-CTLA4Ig, replication-defective adenovirus co-expressing sCD40LIg and CTLA4Ig, was constructed and identified. The co-expression of sCD40LIg and CTLA4Ig was evaluated with confocal laser scanning microscope and Western blotting. Skin transplantations of C57BL/6 to BALB/c mice were performed. PBS, Ad-Shuttle-CMV and Ad-sCD40LIg-IRES(2)-CTLA4Ig were administered. Skin graft survival was monitored and the mRNA expression of both genes was evaluated in the skin allografts.</p><p><b>RESULTS</b>Ad-sCD40LIg-IRES(2)-CTLA4Ig was constructed successfully and identified. The co-expression of sCD40LIg and CTLA4Ig was identified with confocal laser scanning microscopy and Western blotting. Compared to the skin graft mean survival time (MST) of non-treated group ((5.75+/-0.71) d) or Ad-Shuttle-CMV-treated group ((5.50+/-0.53) d), the skin graft MST was dramatically prolonged in the Ad-sCD40LIg-IRES(2)-CTLA4Ig-treated group ((16.38+/-1.19) d, P<0.001). The mRNA expression of both genes was detected.</p><p><b>CONCLUSION</b>Ad-sCD40LIg-IRES(2)-CTLA4Ig, a replication-defective adenovirus carrying genes encoding sCD40LIg and CTLA4Ig, was constructed. Simultaneous blockade of CD40/CD40L and B7/CD28 costimulatory pathway mediated by replication-defective adenovirus significantly prolonged skin allograft survival in mice.</p>